1. Field of the Invention
The present invention relates generally to a method for rapid identification and characterization of binding partners for a target molecule, and for providing modified binding partners with improved binding affinity. More specifically, the invention concerns an improved tethering method for the rapid identification of small molecule fragments that bind near one another on a target molecule. The method is particularly suitable for rapid identification of small molecule ligands that bind weakly near sites of interest through a preformed linker on a target biological molecule (TBM), such as a polypeptide or other macromolecule, to produce higher affinity compounds.
2. Description of the Related Art
The drug discovery process usually begins with massive screening of compound libraries (typically hundreds of thousands of members) to identify modest affinity leads (Kd ˜1 to 10 μM). Although some targets are well suited for this screening process, most are problematic because moderate affinity leads are difficult to obtain. Identifying and subsequently optimizing weaker binding compounds would improve the success rate, but screening at high concentrations is generally impractical because of compound insolubility and assay artifacts. Moreover, the typical screening process does not target specific sites for drug design, only those sites for which a high-throughput assay is available. Finally, many traditional screening methods rely on inhibition assays that are often subject to artifacts caused by reactive chemical species or denaturants.
Erlanson et al., Proc. Nat. Acad Sci. USA 97:9367-9372 (2000), have recently reported a new strategy, called “tethering”, to rapidly and reliably identify small (˜250 Da) soluble drug fragments that bind with low affinity to a specifically targeted site on a protein or other macromolecule, using an intermediary disulfide “tether.” According to this approach, a library of disulfide-containing molecules is allowed to react with a cysteine-containing target protein under partially reducing conditions that promote rapid thiol exchange. If a molecule has even weak affinity for the target protein, the disulfide bond (“tether”) linking the molecule to the target protein will be entropically stabilized. The disulfide-tethered fragments can then be identified by a variety of methods, including mass spectrometry (MS), and their affinity improved by traditional approaches upon removal of the disulfide tether. See also PCT Publication No. WO 00/00823, published on Jan. 6, 2000.
Although the tethering approach of Erlanson et al. represents a significant advance in the rapid identification of small low-affinity ligands, and is a powerful tool for generating drug leads, there is a need for further improved methods to facilitate the rational design of drug candidates.